AN INVESTIGATION OF THE RELATIONSHIP BETWEEN THE eNOS GENE POLYMORPHISM AND DIAGNOSED MIGRAINE
Güler S1,*, Gürkan H2, Tozkir H2, Turan N3, Çelik Y1
*Corresponding Author: Sibel Güler, M.D., Department of Neurology, Trakya University Faculty of Medicine, Balkan Yerleşkesi, 22030 Edirne, Turkey. Tel: +90-284-236-49-81. Fax: +90-284-223-42-03. E-mail: drsibleguler@ yahoo.com
page: 49

MATERIALS AND METHODS

Participants. A total of 300 individuals, comprising 175 migraine patients [25 (14.3%) males, 150 (85.7%) females] and 125 controls [28 (22.4%) males and 97 (77.6%) females] were recruited. Local ethics committee approval was obtained from the Trakya University Medical Faculty Ethics Committee on 13 February 2013, and the official writing number is 2013/26. The participants with migraine had all been referred to the Trakya University Medical Faculty Neurology Policlinic, Edirne, Turkey between January 2013 and December 2013, and had been diagnosed according to the International Classification of Headache Disorders (ICHD-II) criteria [6]. The need to recruit 103 cases to both patient and control groups was calculated on the basis that polymorphisms might be observed as 0.34 in the control group, and an odds ratio (OR) of 2.21, so in order to find an eNOS polymorphism association at 80.0% power, 300 people consisting of 175 patients and 125 controls were recruited. Patients who had cardiovascular, renal, hepatic, gastrointestinal, pulmonary, endocrine, oncologic, autoimmune, respiratory and inflammatory diseases were excluded. Controls were selected randomly among people who had no cardiovascular, renal, hepatic, gastrointestinal, pulmonary, endocrine, oncologic, autoimmune, respiratory, inflammatory and psychiatric diseases. Genotype determination. Peripheral blood samples from both patients and controls were drawn into 2 mL EDTA tubes. DNA isolation from peripheral blood samples were performed using Qiagen DNA isolation kits (EZ1® DNA Blood 200 μL Kit; Qiagen, Hilden, North Rhine-Westphalia, Germany) with an EZ1 Advanced XL (Qiagen) Nucleic Acid Isolation system. Consequently, DNA concentration and purity of isolated DNA samples was measured using a NanoDrop device [NanoDrop 2000C; Thermo Fisher Scientific Inc., Wilmington, MA, USA]. After measurement of concentration and purity, amplification polymerase chain reaction (PCR) for pyro-sequencing was performed according to the manufacturer’s recommended PCR protocol, using a PyroMark PCR kit (Qiagen) and primers in a PyroMark Custom Assay Kit (Qiagen) for detection of each polymorphism, such as rs743506, rs207468799, rs3918226, rs2070744, rs1799983, rs148554851 and rs180079. Amplification PCR performed with initial denaturation at 95 °C for 15 min., followed by 45 cycles at 94 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds, and a final extension 72 °C for 10 min. After PCR amplification, PCR products were pyro-sequenced to detect each polymorphism using sequencing primers from a PyroMark Custom Assay Kit, according to the manufacturer’s instructions (PyroMark Q24 System; Qiagen). The results were then analyzed using the PyroMark Q24 software system (Qiagen), and the genotypes for polymorphisms were determined using samples from both controls and patients (Qiagen). Statistical analyses. Statistical evaluation was performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) (SPSS v21) statistics software. One sample Kolmogorov- Smirnov test was used to assess the eligibility for normal distribution of measured data, and since the data did not show a normal distribution, the Mann Whitney U test and the Kruskal-Wallis analysis of variance were used for comparison between groups. Pearson’s c2 test, Fisher’s exact c2 analysis and the Kolmogorov-Smirnov two-sample test were used to analyze qualitative data. Median (minimum-maximum) values and mean value ± standard deviation (SD) were determined as descriptive statistics. The significance limit was set at p <0.05 for all statistics.



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